1. Field of the Invention
The present invention pertains generally to a receptor protein tyrosine kinase (rPTK) ligand. More particularly, the invention relates to a novel ligand which binds to, and activates, the hepatoma transmembrane kinase (Htk) receptor (also known as HpTK 5 receptor) and the isolation and recombinant production of the same.
2. Description of Related Art
Transduction of signals that regulate cell growth and differentiation is regulated in part by phosphorylation of various cellular proteins. Protein tyrosine kinases are enzymes that catalyze this process. Members of the protein tyrosine kinase family can be recognized by the presence of several conserved amino acid regions in the tyrosine kinase catalytic domain (Hanks et al., Science 241:42-52 [1988]). The tyrosine kinase domain is involved in the signal transduction pathways of mitogenesis, transformation and cell differentiation. Certain tyrosine kinases predominantly stimulate cell growth and differentiation, whereas other tyrosine kinases arrest growth and promote differentiation. Furthermore, depending on the cellular environment in which it is expressed, the same tyrosine kinase may either stimulate, or inhibit, cell proliferation. See Schlessinger et al., Neuron 9:383-391 [1992].
Receptor protein tyrosine kinases (rPTKs) convey extracellular signals to intracellular signaling pathways thereby controlling cell proliferation and differentiation. These rPTKs share a similar architecture, with an intracellular catalytic portion, a transmembrane domain and an extracellular ligand-binding domain. (Schlessinger et al., supra). The extracellular domains (ECDs), which are responsible for ligand binding and transmission of biological signals, have been shown to be composed of a number of distinct structural motifs. The intracellular domain comprises a catalytic protein tyrosine kinase.
Receptor tyrosine kinases are categorized in several classes, according to sequence and structural similarities. For example, Class V receptors have cysteine rich and fibronectin Type III regions in the extracellular domain and include the EPH, ELK, ERK, EEK, ECK and HEK receptors. For a review of the various classes of receptor tyrosine kinases and their functions, see, e.g., Hanks et al., supra and Schlessinger et al., supra.
Protein ligands for receptor protein tyrosine kinases bind to the extracellular domain of their cognate receptors at the cell surface and thereby stimulate tyrosine phosphorylation. Several of these ligands are growth factors or cytokines, such as insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), fibroblast growth factor (FGF), and nerve growth factor (NGF). Ligands for a number of tyrosine kinase receptors have been shown to function within the hematopoietic system. For example, the ligand for murine flt3/flk-2 tyrosine kinase receptor, recently cloned, stimulates the proliferation of primitive mouse hematopoietic cells and human CD34-positive bone marrow cells. Lyman et al., Cell 75:1157-1167 (1993).
A protein ligand which stimulates phosphorylation of the ECK receptor has recently been cloned and expressed in CHO cells. Bartley et al., Nature 368:558-560 (1994). This ECK ligand was found to be identical to B61, a molecule previously isolated by Holzman et al., Mol. Cell. Biol. 10:5830-5838 (1990).
A receptor tyrosine kinase has been recently identified and cloned from a human hepatocellular carcinoma cell line, Hep 3B. This receptor, called "Htk" receptor or "HpTK 5" receptor, is thought to belong to the Class V or EPH subfamily or rPTKs. See Bennett et al., J. Biol. Chem., 269(19):14211-14218 (1994).
Northern blot analysis of human fetal tissues revealed that expression of Htk receptor nucleic acid occurs in heart, lung, liver, brain and kidney. In adult human tissue, no signal was detectable in brain, while placenta had a particularly intense signal followed by kidney, liver, lung and pancreas. Skeletal muscle and heart were of lower signal intensity. See Bennett et al., supra.
Htk receptor nucleic acid expression in human tumor cell lines has also been analyzed by Northern blot analysis. Cell lines derived from liver, breast (MCF-7), colon (Colo 205), lung (NCI 69), melanocyte (HM-1) and cervix (HeLa) had detectable signals of appropriate size. Message was present in select cell lines of hematopoietic origin. K562 (a primitive myeloid cell with multipotential), THP-1 (a monocytoid cell), U937 (a myelomonocytic cell line), Hep3B (a human hepatocarcinoma cell line), and CMK (of megakaryocytic origin) were all positive for Htk receptor message, but lymphoid (H9, Jurkat, JH-1, Raji, Ramos) or select other myeloid cells (KG-1 or KMT2) had no detectable transcript by Northern analysis. See Bennett et al., supra.
The mouse homologue to the Htk receptor, called "myk-1", was isolated from mammary gland epithelia. See Andres et al., Oncogene 9:1461-1467 (1994). Andres et al. report that myk-1 is induced during proliferation of mammary epithelium and down-regulated during its differentiation. Additionally, deregulated expression of the receptor is considered to potentially represent an early event in mammary gland carcinogenesis (see Andres et al., supra).
However, it is believed that the protein ligand for the Htk receptor has not been heretofore disclosed. Therefore, it is an object of the invention to provide a ligand to the Htk receptor.
It is a further object of the invention to provide nucleic acid encoding the Htk ligand so that the ligand can be made by recombinant DNA techniques.
These and other objects will be apparent to the ordinary artisan upon consideration of the specification as a whole.